Development of resistance to chemotherapy and invasion to other secondary sites are common features of solid tumor malignancies. It is well known that development of resistance to chemotherapeutic agents is caused by over-expression of proteins involved in drug and apoptosis resistance. The invasion process is also fairly well documented. Tumor invasion is caused by an increased motility of cancer cells and the expression of genes that cause degradation of the proteins in the extra-cellular matrix. There is growing evidence that resistance to chemotherapy and tumor invasion might share a common starting point through a biological process called the epithelial-to-mesenchymal transition (EMT). Recent studies have demonstrated that transforming growth factor beta (TGFβ) may be a critical mediator of EMT. Despite these advances, few therapeutic avenues are available to inhibit the development of chemo-resistance and the spread of cancer to other organs. It has also emerged in the recent literature that certain tumor cells undergoing EMT dedifferentiate and adopt stem cell-like properties (cancer stem cells or CSCs). As is the case for normal stem cells, CSCs are inherently refractory to chemotherapy and radiation therapy. Therefore, targeting a specific regulator of EMT and CSC maintenance represents a very promising therapeutic strategy to increase response to chemotherapeutic agents and to prevent recurrence of cancer.
Utilizing well characterized cell lines as models of EMT, proteins were identified that were up-regulated upon induction of EMT. One of these, a secreted protein termed clusterin (sCLU), was found to be stimulated during EMT and could, on its own, promote the EMT process (Lenferink et al., 2009). Several high-affinity antibodies were generated that interact with sCLU, which when tested in cell-based assays for their ability to block the EMT, those antibodies that neutralized EMT all bound to the same critical amino acid sequence in the sCLU protein. This discovery demonstrated that a specific region of sCLU was responsible for mediating its EMT-promoting activity. By blocking the EMT-epitope in sCLU, the antibodies, in particular an antibody designated 16B5 could block EMT as exemplified by the maintenance of the membrane expression of the epithelial cell marker, E-cadherin, when incubated with cancer cells. Furthermore, human xenograft animal studies using prostate cancer and pancreatic cancer tumors showed that blocking the activity of tumor-associated sCLU resulted in the increased response to standard chemotherapeutic drugs such as docetaxel and gemcitabine, as measured by a significant reduction in tumor growth. Taken together, these results demonstrated that blocking EMT with an antibody capable of interacting with a specific region in sCLU resulted in tumor growth inhibition and increased response to cytotoxic drugs (see international application No. PCT/CA2006/001505 published under No. WO2007/030930 and international application No. PCT/CA2010/0001882 published under No. WO2011/063523, the entire content of which is incorporated herein by reference
Lung cancer is one of the most common cancers and a leading cause of death worldwide, with over a million cases diagnosed yearly and non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancers. Despite recent improvements in diagnostic and therapeutic approaches, the majority of patients are diagnosed with advanced NSCLC where the median survival remains poor (Adamo et al., 2009).
One of the most important targets in NSCLC is the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, that is a cell membrane receptor that plays an important role in proliferation and survival of cancer cells. It is a large transmembrane glycoprotein that serves as a receptor for EGF and several additional endogenous ligands. It has three domains consisting of an extracellular region, a transmembrane domain and an intracellular tyrosine kinase (TK) domain. Functionally, ligand binding to EGFR induces receptor dimerization leading to a structural change that promotes autophosphorylation and activation of the intracellular TK domain. Consequently, EGFR activation influences multiple downstream signaling pathways, including Ras/Raf/mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3′-kinase (PI3K)/Akt pathway, which influence cell proliferation, invasiveness, motility, survival and apoptosis (Shigematsu et al., 2005).
Although EGFR is ubiquitously expressed, it is often modified in tumors cells. These modifications include gene amplification, overexpression of ligands and/or receptors and activating mutations. Overexpression or dysregulation of EGFR or its primary ligands is characteristic of many solid human tumors, including lung cancer. In NSCLC, between 43 and 83% of tumors overexpress EGFR (Adamo et al., 2009). Several agents against EGFR such as monoclonal antibodies that target the extracellular domain or small molecules are able to inhibit the TK activity.
The status of EGFR in metastatic NSCLC and the response to chemotherapy is the subject of much debate. Despite the high proportion of tumors with increased expression of EGFR, some clinical studies have shown that this was a poor predictor of response in first-line therapy (Barr et al., 2008). Furthermore, despite a mild but significant response in patients treated first-line with cisplatin, vinorelbine and cetuximab (EGFR monoclonal antibody) compared to chemotherapy alone, there was no correlation between cetuximab and EGFR over-expression (Mirshahidi and Hsueh, 2010). Overall, an overwhelming amount of clinical results with EGRF inhibitors in NSCLC showed that the status of the receptor was not important in first-line therapy until a recent study reported results showing that patients with high EGFR expression that were treated with cetuximab and chemotherapy exhibited an increase in overall survival compared to chemotherapy alone (Pirker et al., 2012). It is clear from these results that there are other mechanisms involving EGFR in tumors that influence the response of NSCLC patients to EGFR inhibitors.
Additional characteristics of EGFR that likely influence the response of inhibitors are those that permit the binding of small molecules to the TK activity of the receptor. A few have been developed and approved for cancer indications including gefitinib and erlotinib, two small molecules that mimic ATP-binding to this region thus preventing intracellular signaling. Neither of these inhibitors was found to be active in NSCLC in first-line therapy but significant clinical responses were achieved in second- and third line settings (Mirshahidi and Hsueh, 2010). Interestingly, EGFR overexpression had no influence on patient response but it was discovered that activating mutations in EGFR and certain other genes were critical. For example, activating mutations were found to lead to significant increase in progression free survival in patients treated with gefitinib (Costanzo et al., 2011). In addition, NSCLC patients treated with erlotinib who also had mutations in a gene called KRAS, showed no response (Herbst and Sandler, 2008). Thus, patient selection is critical for attempting to understand if they will be responders to EGFR TK inhibitors.
As described above, EMT can have a tremendous influence on the way tumors cells will respond to therapy and the ability of cancer cells to remain epithelial is critical for this response. In cell-based studies, cells that have increased expression of the epithelial cell marker, E-cadherin, are more sensitive to EGFR inhibitors (Barr et al., 2008). In agreement with these observations, there was a correlation between E-cadherin expression and sensitivity to erlotinib (Yauch et al., 2005). At the tumor level, it has been shown that restoration of E-cadherin expression increases sensitivity to EGFR inhibitors (Witta et al., 2006). To date, however, the link between EMT and EGFR status in clinical trials has not been clearly examined. However, given the lack of correlation between EGFR overexpression and response to EGFR inhibitors and the influence of activating EGFR mutations on their response, it is probable that additional influences such as EMT might be directly involved in increasing the efficacy of EGFR inhibitors in NSCLC patients.
This present application provides a method of treatment with an antibody that blocks EMT by inhibiting sCLU in tumors that express EGFR. The EGFR status in these tumors might include EGFR gene amplifications or amplification in EGFR ligands. Furthermore, the tumors cells might include increased autocrine signaling through EGFR and EGFR protein partners. The EGFR status might also include activating mutations in EGFR that cause the receptor to exhibit increased activity. In another embodiment, the tumors might be sensitive or resistant to EGFR inhibitors, including monoclonal antibodies against EGFR or small molecule inhibitors that abrogate the activity of EGFR. Furthermore, EGFR status might also include tumors that were a priori negative for EGFR expression that have reacquired EGFR.
When NSCLC cell lines that express sCLU are exposed to an anti-clusterin antibody, both the expression and the activation of EGFR are increased. In parallel, by virtue of EMT inhibition by the anti-clusterin antibody, the NSCLC cell lines also show increased E-cadherin expression. Taken together, NSCLC patients treated with a clusterin inhibitor in combination with EGFR inhibitors may show an increased response to the EGFR inhibitors.